PROJECT SUMMARY Maintenance of genomic integrity is fundamental to life. DNA damage occurs spontaneously and ubiquitously from endogenous (e.g., reactive oxygen species) and environmental sources (e.g., ultraviolet (UV) light), inflicting mutagenic and cytotoxic lesions upon the genome that drive the progression of cancer and aging. Cellular excision repair (ER) pathways, including base excision repair (BER) and nucleotide excision repair (NER), are a critical 'first line of defense' responsible for recognizing and removing DNA lesions. The overall objective of this proposal is to understand how ER pathways access DNA lesions that are 'buried' in different types of genomic chromatin. Previous studies have shown that histone post-translational modifications (PTMs) and ATP-dependent chromatin remodelers (ACR) are important for ER of DNA lesions in chromatin. However, it is not known how these chromatin remodeling activities and histone PTMs operate during repair on the diverse spectrum of distinct chromatin types in eukaryotic cells. To address this question, we have developed genome-wide methods to map the formation and repair of UV-induced cyclobutane pyrimidine dimers (CPDs) and methyl methanesulfonate (MMS)-induced N-methylpurine (NMP) base lesions. Our published study and preliminary data indicate that the CPD-seq and NMP-seq methods can be used to map DNA lesions across the yeast and human genomes at single nucleotide resolution. To better understand the genomic roles of ACRs in ER, we will use the CPD-seq and NMP-seq methods to measure repair in yeast and human cells depleted of different classes of ACRs (Aim I). Histone acetylation is an important PTM associated with DNA damage responses. Our preliminary data suggest that the Esa1/TIP60 histone acetyltransferase (HAT) complex plays a novel role in ER. To test this hypothesis, we will characterize the roles of Esa1/TIP60 and other HATs in regulating ER in both yeast and human cells (Aim II). Our preliminary data indicate that histone acetylation activity in cell-free repair extracts is important for repair of base lesions occluded in nucleosomes. These findings provide the foundation of Aim III, which will identify histone PTMs associated with BER, and characterize their functional role in BER of nucleosomes. Finally, we will investigate the detailed molecular mechanisms by which histone acetylation and ACRs regulate the activity of purified BER enzymes on mononucleosome and oligonucleosome substrates in vitro containing 'designed' DNA base lesions (Aim IV). This proposal is an ongoing investigation of the effects of DNA packaging in chromatin on the two major ER pathways (NER and BER) found in cells. As all eukaryotes, including humans, must deal with this `packaging paradox' for surveillance of the genome, results from these studies are relevant to the broad spectrum of cancer etiology, prevention and treatment.